11, 17-oxygenated a-norprogesterones



rates This invention relates to and has as its object the provision of new physiologically active steroids, methods for their production and novel intermediates useful in said preparation.

More particularly, this invention relates to the provision of steroids of the formulae and wherein R and R are selected from the group consisting of hydrogen and acyl.

The preferred acyl radicals are those of hydrocarbon carboxylic acids of less than twelve carbon atoms, as exemplified by the lower alkanoic acids (e.g., acetic, propionic, butyn'c and tert-pentanoic acid), the lower alkenoic acids, the monocyclic aryl carboxylic acids (e.g., benzoic and toluic acids), the monocyclic aryl lower alkanoic acids (e.g., phenacetic and fi-phenylpropionic acid), the cycloalkane carboxylic acids and the cycloalkene carboxylic acids.

The novel compounds of this invention are pharmacologically active substances which possess anti-androgenic activity (i.e., they inhibit the actions of androgens), and which may be used in the treatment of such conditions as hyperandrogenic acne.

The compounds may be formulated for such administration, the concentration and/or dosage being based on the activity of the particular compound and the requirements of the patient.

The final products of the instant invention may be prepared by the processes of this invention which entails a number of steps beginning with 17a-hydroxy-A-norprogesterone as starting material. The preparation of this compound is disclosed in copending US patent application, Ser. No. 399,838, filed Sept. 28, 1964. The process of the instant invention may be represented by the following equations wherein R and R are as hereinbefore defined:

3,33%,861T Patented July 11, 1967 It has been found that a new intermediate compound (Compound II) of this invention, namely, 11a,l7a-dihydroxy-A-norprogesterone can be prepared from the starting material by subjecting the latter to the action of a microorganism of the genus Aspergillus or to the action of the enzymes thereof under oxidizing and preferably aerobic conditions.

To prepare the compounds of this invention, the starting reactant may be first subjected to the action of enzymes of a microorganism of the genus Aspergillus under oxidizing conditions. This oxidation can best be effected either by including the starting reactant in an aerobic culture of the microorganism, or by bringing together an aqueous medium, the compounds, air, and enzymes of non-proliferating cells of the microorganism.

In general, the conditions of culturing the microorganism for the purposes of this invention are (except for the inclusion of the statring reactant to be converted), the same as those of culturing various other microorganisms for the production of antibiotics, and other like substances. The microorganism is grown aerobically in contact with (in or on) suitable fermentation medium. A suitable medium essentially comprises a source of carbon and energy. The latter may be a carbohydrate, for example, molasses, glucose, maltose, starch or dextrin, a fatty acid, a fat and/ or the compound itself. Preferably, however, the medium includes an assimilable source of carbon and energy in addition to the steroid. Among the fats utilizable for the purpose of this invention are lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein and trilaurin. Among the fatty acids utilizable for the purpose of this invention are stearic acid, palmitic acid, oleic acid, linoleic acid and myristic acid.

The source of nitrogenous factors utilizable for the purposes of this invention may be organic (e.g., soybean meal, cornsteep liquor, yeast extract, meat extract and/ or distillers solubles) or synthetic (i.e., composed of simple, synthesizable organic or inorganic compounds, such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation, for example, by the conventional methods of exposing a large surface of the medium to air or by utilizing submerged aerated culture. The compound may be added to the culture during the incubation period, or included in the medium prior to sterilization or inoculation. The preferred (but not limiting) range of the concentration of the compound in the culture is about 0.01% to about 0.1%. The culture period (or rather the time of subjecting the compound to the action of the enzyme) may vary considerably, the range of about twenty-four to ninety-six hours being feasible, but not limiting.

Compound A is then acylated to form new Compounds B as by treatment with an acid anhydride, for example,

acetic anhydride to yield the 11a-acyloxy-17u-hydroxy derivatives. When an excess of the acid anhydride is utilized in the presence of perchloric acid, the 11a,17adiacyloxy derivatives are formed. Alternatively, Compound A may be oxidized directly by treatment with an equivalent amount of an oxidizing agent, such as chromic acid to form the ll-oxo derivatives of Compounds C. This latter compound may then be acylated as set forth above to form the ll-oxo, l7a-acyloxy derivative of Compounds C.

The invention may be illustrated by the following examples, all temperatures are in degrees centigrade unless otherwise stated:

Example 1 .110a,]7oz-dihydroxy-A-n0rpr0gester0ne (A) F ermentatiom4urface growth from each of 2 two-week old agar slants of Aspergillus ochraceus culture NRRL-405 obtained from Northern Utilization Research and Development Division, USDA, Peoria, 111., the slants containing as a nutrient medium (A):

Grams Glucose 10 Yeast extract 2.5 K HPO 1 Agar 20 Distilled water to 1 liter.

is suspended in 5 ml. of 0.01% aqueous sodium lauryl sulfate solution. One ml. portions of this suspension are used to inoculate six 250 ml. Erlenmeyer flasks, each containing 50 ml. of the following sterilized medium (B):

Grams Cornsteep liquor 40 Glucose 20 Distilled water to 1 liter.

(Adjust to pH 6.5 with NaOH before sterilization.)

After seventy-two hours incubation at 25 C. with continuous rotary agitation (280 cycles/minute; two inch radius), 5% (vol/vol.) transfers are made to thirtyfour 250 ml. Erlenmeyer flasks each containing 50 ml. of the following sterilized medium (C):

Grams Cornsteep liquor 1O Glucose 5 Distilled water to 1 liter.

(Adjust to pH 6.5 with NaOH before sterilization.)

After twenty-four hours incubation, using the same conditions as described above, the steroid (300 micrograms/ml.) is added by supplementing each flask with 0.25 ml. of a sterile solution (60 mg./ml.) of 17a-hydroxy A norprogesterone in N,N-dimethylformamide. A total of 510 mg. is fermented. After five days of further incubation, using identical conditions as described above, the contents of the flasks are pooled and filtered through a Seitz clarifying pad. The flasks, mycelium and pad are washed with successive 50 ml. portions of warm water. The combined filtrate and washings have a volume of (B) Isolation-The thus obtained filtrate is extracted three times with 600 ml. portions of chloroform. The chloroform extracts are washed three times with 600 ml. portions of water, dried over sodium sulfate, and evaporated in vacuo to give a 452 mg. residue. The residue is plate chromatographed using neutral alumina (Activity V) as the adsorbent and chloroform containing 2% methanol as the developing solvent. Elution of the most polar ultraviolet absorbing band with warm ethyl acetate, and crystallization of the residue from acetonitrile gives 114 mg. of 11a,17u-dihydroxy-A-norprogesterone having a melting point of 230232. The analytical sample is prepared by recrystallization from acetonitrile, M.P. 230-232"; +l2 (EtOH); A 2.89, 5.92, 5.98 and 618 A212 232 my. (15,800)

4 TSKCHBM 9.20 (s, 18-Me), 8.74 (s, 19-Me), 7.72 (s, 21- Me), 7.28 (s, 17-hydroxy), 6.08 (b, llfi-H) and 4.18 (s, 3-H).

Analysis.Calcd for Cz HzgOg (332.42): C, 72.26; H, 8.49. Found: C, 72.25; H, 8.48.

Example 2.-11u-acet0xy-1 7a-hydr0xy-A- norprogesierone A mixture of 30 mg. of l1a,17a-dihydroxy-A-norprogesterone, 0.2 ml. of acetic anhydride and 0.4 ml. of pyridine is left at room temperature for four hours, diluted with ice-water and the precipitate collected by filtration to give 27 mg. of 1la-acetoxy-17a-hydroxy-A- norprogesterone having a melting point of 275277 (d). The analytical sample is prepared by recrystallization from chloroform-isopropyl ether, M.P. 275277; [(11 +2l (EtOH); A 2.88, 5.78, 5.87 and 6-19/l;

TSKCHM 9.13 (s, 18-Me), 8.82 (s, 19-Me), 7.93 (s, 11- acetate), 7.73 (s, 2l-Me), 4.75 (m, llfi-H) and 4.20 (s, 3-H).

Analysis.Calcd for C H O (374.46): C, 70.56; H, 8.08. Found: C, 70.42; H, 8.24.

Example 3.11nc,1 7u-dz'acet0xy-A-norpr0gesterone Example 4.11-kei0-1 7a-lzydroxy-A-n0rprogester0ne A solution of 30 mg. of 11a,17u-dihydroXy-A-norprogesterone in 5 ml. of acetone is treated dropwise with an equivalent amount of chromium trioxide-sulfuric acid. The chromic sulfate is removed by filtration, and washed with additional acetone. The filtrate is concentrated, diluted with water and extracted with chloroform. The chloroform extracts are washed with 8% salt solution, dried over sodium sulfate and evaporated to dryness. Crystallization of the residue from methanol-isopropyl ether gives 12 mg. of 1l-keto-17a-hydroxy-A-norprogesterone having a melting point of 267-269". The analytical sample is prepared by recrystallization from acetonitrileisopropyl ether, M.P. 270.5272.5, A 2.92, 5.88, 5.98 and 6.17;; )t 228 m (15,400).

Analysis.Calcd for C H Q, (330.41): C, 72.70; H, 7.93. Found: C, 72.75; H, 7.85.

Example 5.] 1 -ket0-1 7 a-acetoxy-A -norprogesterone Following the procedure in Example 3 but substituting 11-keto-17a-hydroxy A norprogesterone for 11a,17a-dihydroxy-A-norprogesterone there is obtained 11-keto-17aacetoxy-A-norprogesterone.

While there have been described various embodiments of the invention, the compositions and methods described are not intended to be understood as limiting the scope of the invention, as it is realized that changes therein are possible and it is further intended that each element recited in any of the following claims is to be understood as referring to all equivalent elements for accomplishing substantially the same results in substantially the same or equivalent manner, itcovering the invention broadly in whatever form its principle may be utilized.

5 6 What is claimed is: of hydrogen and acyl wherein the acyl is from a hydro- 1. A compound selected from the group consisting of carbon carboxylic acid of less than twelve carbons. steroids of the formulae 2. 11a;17a-dihydroxy-A-norprogesterone.

3. 11a-acetoxy-l7u-hydroxy-A-norprogesterone. CH3 CH 5 4. 11a,17u-diacetoxy-A-norprogesterone. (3:0 5. 11-keto-l7a-hydroXy-A-norprogesterone. I 6. 1l-keto-17a-acetoxy-A-norprogesterone. R0 0 References Cited 10 UNITED STATES PATENTS O=/\ 3,165,541 1/1965 Hydorn et a1. 260586 and 3,170,919 2/1965 Fried 260-586 LORRAINE A. WEINBERGER, Primary Examiner. wherein R and R are selected from the group consisting 15 V. GARNER, Assistant Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF STEROIDS OF THE FORMULAE 